Obstructive pulmonary diseases such as asthma and COPD are frequently treated with -agonists. These drugs target the 2AR expressed on airway smooth muscle and are used for both long term control and rescue from acute bronchospasm. Of particular importance in human pathophysiology and treatment, 2AR signaling can be desensitized by b-agonist treatment, as well as the asthmatic inflammatory milieu. The mechanisms of this regulation are poorly understood, particularly in smooth muscle with associated physiologic function. These gaps in our knowledge have impaired our basic understanding of receptor-relaxation coupling and our ability to improve therapy for the treatment of bronchospasm. Mechanisms which will be explored in this grant include regulation by protein kinase A (PKA) and G-protein coupled receptor kinases (GRKs), and the let-7 family of microRNAs (miRNA). Our previous studies in airway smooth muscle point to a significant role for PKA phosphorylation of the receptor that alters GRK interaction with 2AR which had not been previously defined. Specific Aim 1 will delineate this mechanism of altered 2AR function with studies addressing phosphorylation, 2-arrestin recruitment, receptor internalization and phosphodiesterase (PDE) recruitment. Additionally, we have identified that 2AR baseline expression is directly regulated by let-7 miRNA. And, in an apparent feedback loop, let-7 miRNA is regulated by prolonged agonist activation of 2AR. In Specific Aim 2, transgenic mice will be generated with targeted expression on airway smooth muscle of WT 2AR and a mutant 2AR lacking the let-7 seed region in the 3UTR. These mice will be studied to ascertain the significance of let-7 in establishing 2AR expression and its physiologic relevance in regulating bronchodilation. Additional studies will focus on the mechanisms and physiologic relevance of the apparent feedback loop, which may establish a new, and perhaps the most important, mechanism of 2AR downregulation by agonist. Loss of 2AR function in the asthmatic state has been reported in humans and animal models yet the mechanisms remain unknown, particularly with regard to linking cellular events to physiologic consequences. Evidence suggests several potential mechanisms that may involve PKA and GRK actions at the receptor, or altered let-7 miRNA expression. We have developed several transgenic mice that express mutated 2AR so that these potential mechanisms can be explored. In Specific Aim 3 transgenic mice lacking phosphorylation sites for PKA, GRK, PKA/GRK and the let-7 binding domains will be crossed into the inducible IL-13 allergic mouse model with confirmatory studies in the ovalbumin (OVA) allergen induced mouse model. Results from these various transgenic mice will define the specific mechanisms relevant to 2AR dysfunction in the asthmatic state. Taken together, these proposed studies will combine molecular, cellular and transgenic approaches to define mechanisms of 2AR function and regulation relevant to asthma.